title: A multifunctional lentiviral-based gene knockdown with concurrent rescue that controls for off-target effects of RNAi.
abstract: The efficient, stable delivery of siRNA into cells, and the appropriate controls for non-specific off-target effects of siRNA are major limitations to functional studies using siRNA technology. To overcome these drawbacks, we have developed a single lentiviral vector that can concurrently deplete endogenous gene expression while expressing an epitope-tagged siRNA-resistant target gene in the same cell. To demonstrate the functional utility of this system, we performed RNAi-depleted α-actinin-1 (α-ACTNl) expression in human T cells. α-ACTNl RNAi resulted in inhibited chemotaxis to SDF-lα, but it can be completely rescued by concurrent expression of RNAi-resistant α-ACTNl (rr-α-ACTNl) in the same cell. The presence of a GFP tag on rr-α-ACTNl allowed for detection of appropriate subcellular localization of rr-α-ACTNl. This system provides not only an internal control for RNAi off-target effects, but also the potential tool for rapid structure-function analyses and gene therapy.
authors.affiliation: Department of Medicine, Washington University, St. Louis, MO 63110, USA.
authors.forename: Yunfeng
authors.initials: Y
authors.lastname: Feng
authors.forename: Linghu
authors.initials: L
authors.lastname: Nie
authors.forename: Meghna Das
authors.initials: MD
authors.lastname: Thakur
authors.forename: Qin
authors.initials: Q
authors.lastname: Su
authors.forename: Zhenfen
authors.initials: Z
authors.lastname: Chi
authors.forename: Yongliang
authors.initials: Y
authors.lastname: Zhao
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Longmore
mesh_terms: D000185:Actinin / Q000235:genetics; D002460:Cell Line; D054443:Cell Migration Assays; D054377:Chemokine CXCL12 / Q000378:metabolism; D055785:Gene Knockdown Techniques / Q000379:methods*; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D009690:Nucleic Acid Conformation; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D013601:T-Lymphocytes / Q000378:metabolism
chemical_list: C526905:ACTN1 protein, human; C515255:CXCL12 protein, human; D054377:Chemokine CXCL12; D034741:RNA, Small Interfering; D000185:Actinin; D049452:Green Fluorescent Proteins